Structural Determinants of Synaptobrevin 2 Function in Synaptic Vesicle Fusion

Home , SNARE (protein), Synaptobrevin, Syntaxin, Vesicle fusion

6668 • The Journal of Neuroscience, June 21, 2006 • 26(25):6668–6676

Cellular/Molecular Structural Determinants of Synaptobrevin 2 Function in Synaptic Vesicle Fusion

Ferenc Dea´k,1,2 Ok-Ho Shin,1 Ege T. Kavalali,1,4 and Thomas C. Su¨dhof 1,2,3 1Center for Basic Neuroscience, 2Howard Hughes Medical Institute, Departments of 3Molecular Genetics and 4Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111

Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachmentproteinreceptor(R-SNARE),severelydecreasesbutdoesnotabolishspontaneousandevokedsynapticvesicleexocytosis.We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca 2ϩ-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca 2ϩ-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically. Key words: exocytosis; endocytosis; synaptic vesicle recycling; SNARE; spontaneous fusion; hippocampus; synapse; whole-cell patch-clamp

Introduction bled as four-helical bundles composed of four SNARE motifs (in Synapses release neurotransmitter when an action potential (AP) the case of synaptic SNARE complexes, a single SNARE motif induces Ca 2ϩ influx into nerve terminals, and Ca 2ϩ in turn trig- each from synaptobrevin and syntaxin and two from SNAP-25) gers synaptic vesicle exocytosis. However, quantal vesicular re- (Hanson et al., 1997). Assembly of SNARE complexes is thought lease that is electrically indistinguishable from AP-driven release to catalyze fusion by forcing membranes into close proximity also occurs spontaneously in all synapses; moreover, under ex- (Weber et al., 1998). Although SNARE complex formation is perimental conditions, release can be induced by hypertonicity. primarily fueled by hydrophobic interactions, SNARE complexes Although all of these forms of release require soluble contain a central “zero layer” composed of a hydrophilic electro- N-ethylmaleimide-sensitive factor attachment protein receptor static interaction mediated by three glutamine and one arginine (SNARE) proteins (for review, see Jahn et al., 2003), it is currently residue (Sutton et al., 1998). As a result, SNARE proteins are unclear whether the different forms of release are mechanistically classified based on their SNARE motif sequences into types of identical or are mediated by distinct types of fusion. Q-SNAREs and R-SNAREs (Fasshauer et al., 1998). During synaptic vesicle fusion, the synaptic vesicle SNARE Synaptobrevin 2 is a minimal SNARE that consists only of a protein synaptobrevin 2 (Syb2)/vesicle-associated membrane short NH2-terminal sequence, a SNARE motif, and a COOH- protein 2 (VAMP2) forms a complex with the plasma membrane terminal transmembrane region (see Fig. 1A). Initial evidence SNARE proteins syntaxin 1 and synaptosome-associated protein that synaptobrevin mediates fusion was obtained when synapto- of 25 kDa (SNAP-25) (Sollner et al., 1993). All SNARE proteins brevin was identified as the target of tetanus toxin, which blocks contain one or two SNARE motifs; SNARE complexes are assem- exocytosis by proteolytic cleavage of synaptobrevin (Link et al., 1992; Schiavo et al., 1992). Deletion of synaptobrevin impairs all Received Dec. 9, 2005; revised May 3, 2006; accepted May 3, 2006. forms of neurotransmitter release, albeit to different degrees This work was supported by the National Institutes of Mental Health Grant MH066198 (E.T.K) and the Howard (Schoch et al., 2001). Synaptobrevin-deficient synapses show an Hughes Medical Institute (F.D., T.C.S.). We are grateful to J. Pessin (Stony Brook, New York) for providing the ϳ100-fold reduction in AP-induced neurotransmitter release cellubrevinknock-outanimals.Wethank,fortheexcellenttechnicalassistance,AndreaRoth,IzaKornblum,andEwa ϳ Borowicz,andNickyHamlinandJasonMitchellforthehelpwithmousehusbandry.Wethankthehelpandadviceof but only a 10-fold reduction in spontaneous release or in re- Weiping Han, Peter Bronk, and Wen-Pin Chang with the lentivirus production. lease triggered by hypertonic sucrose. This disparity between CorrespondenceshouldbeaddressedtoDr.ThomasC.Su¨dhoforDr.EgeT.Kavalali,CenterforBasicNeuroscience, evoked and spontaneous release was also observed in neurons University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9111. E-mail: lacking SNAP-25 (Washbourne et al., 2002). Interestingly, in [email protected] and [email protected]. DOI:10.1523/JNEUROSCI.5272-05.2006 synaptobrevin-deficient chromaffin cells, release was only de- Copyright © 2006 Society for Neuroscience 0270-6474/06/266668-09$15.00/0 creased by ϳ50%, whereas double-mutant mice that lack both Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion J. Neurosci., June 21, 2006 • 26(25):6668–6676 • 6669

synaptobrevin and cellubrevin, a closely related R-SNARE pro- Synaptic boutons were loaded with N-(3-triethylammoniumpropyl)-4- tein (McMahon et al., 1993), lacked all release (Borisovska et al., (4-(diethylamino)styryl)pyridinium dibromide (FM2-10) (400 ␮M; In- ϩ 2ϩ 2005). Thus, in chromaffin cells, synaptobrevin and cellubrevin vitrogen, Carlsbad, CA) by a 90 s exposure to 47 mM K /2 mM Ca ; full are functionally redundant. To some degree, redundancy was destaining was triggered with a hyperkalemic bath solution containing 90 mM KCl substituted for NaCl. Images were taken after 10 min wash in also reported for SNAP-25 and SNAP-23 function in chromaffin ϩ dye-free, nominally Ca 2 -free solution to minimize spontaneous re- cells (Sorensen et al., 2003). lease. In all experiments, isolated boutons (ϳ1 ␮m 2) were selected for Although synaptic SNARE proteins have been extensively analysis. Synaptic vesicle fusion was induced by gravity perfusion of hy- studied biochemically and physiologically, little is known about perkalemic solution onto the field of interest (1–2 ml/min) for 60 s, the structure/function relationships of synaptobrevin. For exam- followed by nominal Ca 2ϩ-free Tyrode’s perfusion for 60 s again. Images ple, the current model for how SNAREs catalyze fusion requires were acquired with a cooled CCD camera (Roper Scientific, Trenton, NJ) that the SNARE motif must be close to the transmembrane re- during illumination (1 Hz and 200 ms) at 480 Ϯ 20 nm (505 dichroic gion, but this has only been examined in yeast fusion reactions long pass and 534 Ϯ 25 bandpass) via an optical switch (Sutter Instru- (Wang et al., 2001) and in in vitro fusion assays (McNew et al., ments, Novato, CA) and analyzed using Metafluor Software (Universal Imaging, Downingtown, PA). Background staining levels determined 1999; Parlati et al., 2000; Melia et al., 2002). Therefore, in the ϩ present study, we used rescue experiments in cultured after five consecutive rounds of high K application were subtracted from all fluorescence images. synaptobrevin-deficient neurons to examine the structural re- Electrophysiology. Synaptic responses were recorded from pyramidal quirements for synaptobrevin function in synaptic exocytosis. cells in modified Tyrode’s bath solution in the whole-cell patch configuration. Data were acquired with an Axopatch 200B amplifier and Materials and Methods Clampex 8.0 software (Molecular Devices, Union City, CA), filtered at 2 ␮ Hippocampal cultures. Double knock-out (KO) mice were bred by cross- kHz, and sampled at 200 s. The internal pipette solution included the ing heterozygous mutant synaptobrevin 2 KO mice (Schoch et al., 2001) following (in mM): 115 Cs-MeSO3, 10 CsCl, 5 NaCl, 0.1 CaCl2,10 that are also homozygous mutant for cellubrevin (Yang et al., 2001). HEPES, 4 Cs-BAPTA, 20 tetraethylammonium-Cl, 4 Mg-ATP, 0.3 mM Hippocampal neurons from embryonic day 18 littermate mice were dis- Na2-GTP, and 10 lidocaine N-ethyl-bromide, pH 7.35 (300 mOsm). A sociated by trypsin (5 mg/ml for 5 min at 37°C), triturated with a sili- hypertonic solution, prepared by addition of 500 mM sucrose to the 2ϩ conized Pasteur pipette, and plated onto 12 mm coverslips coated with nominally Ca -free Tyrode’s solution, was perfused directly into the poly-L-lysine (approximately three coverslips per hippocampus). Neu- close vicinity of the cell from which the recording was made. Field stim- rons were cultured at 37°C in a humidified incubator with 95% air/5% ulations (24 mA pulses of 0.6 ms) were applied with parallel platinum electrodes immersed into the perfusion chamber. CO2 in Minimal Essential Media containing 5 g/liter glucose, 0.1 g/liter transferrin, 0.25 g/liter insulin, 0.3 g/liter glutamine, 5–10% heat- Coimmunoprecipitation of recombinant synaptobrevin 2 mutants with inactivated FCS, 2% B-27 supplement, and 1–2 ␮M cytosine arabinoside, syntaxin 1. Neuronal cultures in a six-well plate were infected with dif- and used after 12–22 d in vitro. ferent synaptobrevin 2 mutant constructs and harvested using 1 ml of a Constructs. The different v-SNARE constructs were assembled in lit- HEPES buffer (50 mM HEPES, pH 7.2, 100 mM NaCl, 2 mM MgCl2,4mM ␮ ␮ mus28 vector (New England Biolabs, Ipswich, MA) and tagged N termi- EGTA, 1 mM PMSF, 5 g/ml leupeptin, 2 g/ml aprotinin, and 1 mM nally with enhanced cyan fluorescent protein (eCFP) lacking the stop codon localizing the fluorescent protein in the cytoplasm. The following primer pairs were used for creating the mutants by PCR reaction: DF0007(C GTA GAC AAG GTC CTC GAG CAG GAC CAG AAG CTG TCG G) and DF0008 (C CGA CAG CTT CTG GTC CTG CTC GAG GAC CTT GTC TAC G) for R56Q point mutation, and P1 (GCT TCG AAT TCT GCA GTC G) and P2 (AAA GGA TCC AGC GCT ACC GGC TGA ACC AGC GCT ACC GGC CTT GAG CTT GGC TGC ACT AGT TTC AAA CTG GG) for the 12 insertion after introducing a new BamHI site with the primer pair of P3 (AAA GGA TCC CGC AAA TAC TGG TGG AAA AAC C) and P4 (CGC GCT AGC TCC TCA GGG GTT TAA GAG C). For the 24-insertion mutant, a digestion with BamHI/NheI was performed on the 12-insertion mutant, and the PCR product for 12 insertion with P4 and P5 (CA AGA TCT GCC GGT AGC GCT GGT TCA GC) was digested with BglII and NheI and subcloned, resulting in an insertion doubled in size. Bacterial expression was performed in Escherichia coli strain BL21. Later, all constructs were transferred into the main lentivirus vector (FUW, technique of lentiviral system was adopted from Dittgen et al., 2004) using BsiWI and EcoRI sites, except for the synaptobrevin 2–24 insertion, which was inserted between XbaI and NheI sites. All constructs were verified by sequencing. For the 12- and 24-insertion constructs, we preferred AGS repeats because of their hydrophobicity and predicted inability to form special secondary structure. Lentiviral infection. Constructs were cotransfected with plasmids for viral envelope proteins and enzymes into HEK 293 cells using the Fu- GENE6 transfection system (Roche Molecular Biochemicals, Indianap- Figure 1. Both N- and C-terminally tagged synaptobrevin 2 rescues synaptic function in olis, IN) according to the specifications of the manufacturer, and synaptobrevin2knock-outneurons.A,Schematicdrawingsforsynaptobrevin2taggedeitherN lentivirus-containing culture medium was harvested 3 d later, filtered terminallywitheCFP(eCFP–Syb2)orCterminallywithanotherGFPanalogfluorescenceprotein through 0.45 ␮m pores, and immediately used for infection or frozen in called venus (Syb2–venus). TMR, Transmembrane region. B, Representative responses to field liquid nitrogen and stored at Ϫ80°C. Hippocampal cultures were in- stimulation at 1 Hz from WT, Syb2 KO hippocampal neurons, or synaptobrevin 2 knock-out fected at day 4 in vitro by adding 400 ␮l of viral suspension to each well. neuronsinfectedwithlentiviruscontainingeCFP–Syb2orSyb2–venus.C,Bargraphdepictsthe Fluorescence imaging. Modified Tyrode’s bath solution [in mM: 150 amplitudes of average responses to the first 60 pulses (n ϭ 6 for WT, Syb2 KO, and Syb2-venus ϭ NaCl, 4 KCl, 2MgCl2, 10 glucose, 10 HEPES-NaOH, pH 7.4, 0.01 CNQX, infected cells; n 5 for eCFP–Syb2 infected cells). Note that statistical significance is marked ϳ Ͻ Ͻ Ͻ 0.05 AP-5, and 2 CaCl2 ( 310 mOsm)] was used in all experiments. on this and following figures as follows: *p 0.05, **p 0.01, ***p 0.001. 6670 • J. Neurosci., June 21, 2006 • 26(25):6668–6676 Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion

DTT). Proteins were extracted using 1% Triton X-100 for 1.5 h with rotation at 4°C and centrifuged at 20,800 ϫ g for 10 min, and superna- tants were diluted with the same HEPES buffer without Triton X-100 to reduce Triton X-100 concentration to 0.5%. A total of 10 ␮l of rabbit serum (U6251) raised against rat syntaxin 1 residue 1–264 was added to 600 ␮l of 0.5% Triton X-100 cell lysate and incubated for2hat4°Cwith rotation. After 2 h incubation with U6251, 20 ␮l of protein A-Sepharose beads were added and further incubated another 1 h with rotation at 4°C. Beads were washed five times with HEPES buffer containing 0.5% Triton X-100, and 35 ␮lof2ϫ SDS sample buffer was added, vortexed, and boiled for 10 min. Immunoprecipitation sample preparations (30 ␮l) were used for SDS-PAGE and Western blotting. A total of 25 ␮l of cell lysate was used for SDS-PAGE and Western blotting after trichloroacetic acid precipitation of total protein. Blots were performed using the following sera or ascites: mouse anti-green fluorescent protein (GFP), C163 from Zymed (South San Francisco, CA); mouse anti-Syb2, CL69.1; mouse anti-syntaxin 1, HPC-1; and mouse anti-SNAP-25, CL71.1. Miscellaneous. Paired Student’s t test or variance analysis was used to determine significance, which is marked on the figures as asterisks: *p Ͻ 0.05, **p Ͻ 0.01, ***p Ͻ 0.001 levels of significance. Results N- or C-terminal GFP fusions of synaptobrevin do not impair its function Because in synaptotagmin 1 fusion of the cytoplasmic C terminus with GFP inactivates function (Han et al., 2005), we tested whether fusion of synaptobrevin with a GFP derivative (to visu- alize expression) at either the N or C terminus impairs function. In these experiments, we used cultured hippocampal neurons from synaptobrevin-deficient mouse embryos and expressed exogenous synaptobrevin proteins using a lentiviral expression system (Dittgen et al., 2004). The main advantage of the lentiviral expression system is its high infection efficacy (Ͼ90%), thus al- lowing electrophysiological assessment of presynaptic function in combination with optical analysis of synaptic vesicle recycling using the fluorescent styryl dye FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide]. Expression of synaptobrevin containing an N-terminally fused or a C-terminally fused GFP derivative (Fig. 1A) fully rescued the amplitude of evoked responses in synaptobrevin-deficient neurons (Fig. 1B). Thus, the GFP moiety does not interfere with synaptobrevin function during exocytosis when attached to synaptobrevin either N or C terminally.

Cellubrevin fully rescues synaptic transmission in synaptobrevin-deficient neurons The R-SNARE protein cellubrevin (VAMP3) is highly homologous to synaptobrevin 2 (McMahon et al., 1993), with only a single amino acid difference between their SNARE motifs, although their N- and C-terminal sequences differ significantly (Fig. 2A). We used eCFP-tagged cellubrevin to test whether cel- Figure2. Rescueofsynaptictransmissioninsynaptobrevin2-deficientsynapsesbycellubre- vin.Synaptictransmissionwasanalyzedafterinfectionofsynaptobrevin2-deficienthippocam- lubrevin can substitute functionally for synaptobrevin. In palcultureswithlentiviruscontainingeithersynaptobrevin2orcellubrevin.A,Schematicdraw- synaptobrevin-deficient neurons, eCFP-tagged cellubrevin was ingscomparethestructureofsynaptobrevin2andcellubrevin.TMR,Transmembraneregion.B, targeted to synapses and fully restored spontaneous neurotrans- Representative traces of spontaneous synaptic release at Ϫ70 mV in the presence of 1 ␮M mitter release, evoked release in response to hypertonic sucrose tetrodotoxin and 100 ␮M picrotoxin. C, Graph depicts the average frequency and SEM of min- application, and evoked release in response to action potentials iature EPSCs (mEPSCs). Note the markedly reduced average frequency of miniature EPSCs for synaptobrevin 2 knock-outs and its rescue by both synaptobrevin 2 and cellubrevin [n ϭ 12 for 4 WT; n ϭ 6 for synaptobrevin 2 knock-out and cellubrevin infected (iCeb); n ϭ 11 for synapto- ϩ brevin 2 infected (iSyb2)]. D, Synapses were loaded with 47 mM K for 90 s in the presence of Readily releasable pool recorded by addition of 500 mOsm sucrose to the bath solution. Repre- 200␮M FM2-10,washedwithcalcium-freeTyrode’ssolution,anddestainedbyapplying90mM sentative traces recorded at Ϫ70 mV. G, Bar graph shows synaptic release rate estimated from K ϩ for 60 s, which was repeated five times with a wash of 60 s after each. Averaged traces of the charge transfer rate [n ϭ 4 for WT, synaptobrevin 2 KO, and synaptobrevin 2 infected ϩ FM2-10 fluorescence during the first round of 90 mM K destaining from wild-type (n ϭ 7), (iSyb2); n ϭ 5 for cellubrevin infected (iCeb)]. H, Representative responses to field stimulation Syb2 KO (n ϭ 5), and synaptobrevin 2 knock-out synapses infected with synaptobrevin 2 (n ϭ at 1 Hz. I, Bar graphs depict the average responses to the first 60 pulses [n ϭ 7 for WT and 7)orcellubrevin(nϭ4).E,Bargraphdepictstherecyclingsynapticvesiclepool,whichcouldbe synaptobrevin2KO;nϭ17forsynaptobrevin2infected(iSyb2);nϭ5forcellubrevininfected ϩ ϩ labeled by 47 mM K for 90 s and destained by five times application of 90 mM K for 1 min. F, (iCeb)]. Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion J. Neurosci., June 21, 2006 • 26(25):6668–6676 • 6671

(Fig. 2) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Cellubrevin can thus mediate the function of synaptobrevin in exocytosis. We next investigated whether cellubrevin can also rescue the synaptic vesicle endocytosis defect observed in synaptobrevin- deficient synapses (Dea´k et al., 2004). A hallmark of synaptobrevin-deficient synapses is a markedly reduced uptake of fluorescent styryl dyes such as FM2-10 and FM1-43 during a ϩ ϩ single round of high K stimulation (47 mM K for 90 s), which reflects a reduction in fast synaptic vesicle endocytosis (Dea´k et al., 2004). The N-terminal region of synaptobrevin could poten- tially mediate its role in fast endocytosis, for instance by trigger- ing nucleation of an endocytic coat. Thus, the absence of this region in cellubrevin may result in diminished endocytic trafficking in cellubrevin-rescued synapses. In contrast to this expecta- tion, the rescue of synaptic transmission by cellubrevin extended to styryl dye uptake and release (Fig. 2D,E). Cellubrevin-rescued synapses took up and released FM2-10 dye with the same effi- ciency as synaptobrevin-rescued synapses, suggesting that the N-terminal region of synaptobrevin (which is different from that of cellubrevin) (Fig. 2A) is not essential for endocytosis. In addition, in cellubrevin-rescued synapses, the rate of FM2-10 loss in ϩ response to 90 mM K stimulation was comparable with wild- type (WT) and synaptobrevin-rescued synapses, indicating full recovery of synaptic vesicle priming. Here, it is important to note that infection of synaptobrevin-deficient neurons with eCFP- tagged synaptobrevin 2 and cellubrevin constructs could only rescue the recycling pool size up to 60–70% of the wild type (Fig. 2E) (see Fig. 4E). There are two possibilities that may account for this observation. First, infections were performed 4 d after plating the cells, so it is likely that a significant portion of synaptic vesicles still lacked synaptobrevin 2 (or cellubrevin) at the time of the experiments. Second, all of these constructs had a N-terminal eCFP tag, which might interfere with other members of the release machinery by either masking a part of synaptobrevin 2 protein or weak dimerization.

Additional deletion of cellubrevin does not aggravate the synaptobrevin knock-out phenotype Because cellubrevin can substitute for synaptobrevin in synaptic Figure 3. Analysis of synaptobrevin 2/cellubrevin double knock-outs. A, Spontaneous syn- exocytosis similar to chromaffin cell exocytosis (Borisovska et al., aptic responses (“minis”) in cultured hippocampal neurons from mutant and control mice. 2005), we tested whether low levels of cellubrevin, below the mEPSCs, Miniature EPSCs. Frequency (B) and amplitude (C) of spontaneous synaptic events. All detection limit of the previous immunoblot analyses (Schoch et data shown are means Ϯ SEMs [n ϭ 9 Syb2 KO, WT, and cellubrevin KO (Ceb KO); n ϭ 14 al., 2001), account for the residual fusion in synaptobrevin- synaptobrevin/cellubrevin double KO mice (SC DKO)]; differences between synaptobrevin sin- deficient synapses. To address this question, we generated and gle KO neurons and synaptobrevin/cellubrevin double KO neurons were not significant. D, Av- analyzed double knock-out mice for synaptobrevin 2 and cel- erage destaining curves for synaptobrevin 2 KO (n ϭ 10, 632 synapses), SC double knock-out lubrevin (see Materials and Methods). We found, however, that synapses (n ϭ 7, 444 synapses), cellubrevin KO (n ϭ 6, 467 synapses), and WT (n ϭ 6, 435 ϩ synapses) neurons during the first 90 mM K application as shown on the top. E, Comparable the amount of residual fusion present in synaptobrevin-deficient pool size of functional vesicles in synaptobrevin 2 KO and SC double KO synapses. Graph depicts synapses was not decreased further in the double knock-out neu- ϩ the recycling synaptic vesicle pool size loaded by a single round of 47 mM K stimulation. A.U., rons (Fig. 3). We detected no additional decrease in mini fre- Arbitrary units. F, Synaptic responses to stimulation with hypertonic sucrose. Representative quency or amplitude (Fig. 3B,C) and no change in the synaptic recordings of synaptic responses to hypertonic sucrose (ϩ0.5 Osm). Traces were obtained vesicle pools monitored by loading synapses with the fluorescent during whole-cell recordings from neurons cultured from synaptobrevin 2/cellubrevin double dye FM2-10 (Fig. 3D,E). Moreover, the response to hypertonic KO mice and cellubrevin KO. G, Summary graph of synaptic responses to hypertonic sucrose applicationmonitoredinWTandvarioussingleanddoubleKOneurons.Abbreviationsusedare sucrose (0.5 M sucrose in Tyrode’s solution) was indistinguish- thesameasinA.Responsesarecalculatedasthecumulativechargetransferintegratedover1s able between synaptobrevin single knock-out neurons and syn- interval at the peak of the response. Data shown are means Ϯ SEMs (n ϭ 8 for synaptobrevin aptobrevin/cellubrevin double knock-out neurons (Fig. 3F,G). 2 KO and WT; n ϭ 7 for SC double KO; n ϭ 5 for cellubrevin KO). These data strongly argue that, in contrast to chromaffin cells (Borisovska et al., 2005), cellubrevin does not have a physiological role in exocytosis from hippocampal synapses. 6672 • J. Neurosci., June 21, 2006 • 26(25):6668–6676 Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion

The Q/R substitution in the synaptobrevin SNARE motif does not impair neurotransmitter release SNARE complex assembly requires the interaction of the three Q- and one R-SNARE motif, with the Q-SNAREs contributing glutamines and the R-SNARE contributing arginine to the central ionic zero layer of the complex. To test the functional significance of this interaction in the synapse, we infected synapses with synaptobrevin 2 mutants with a glutamine instead of an arginine in the central layer (R56Q), thus converting synaptobrevin 2 from an R-SNARE to a Q-SNARE (Fig. 4A). Expression of “Q- synaptobrevin” in synaptobrevin-deficient neurons fully rescued synaptic transmission, including spontaneous neurotransmitter release, release evoked in response to hypertonic sucrose application, and release evoked by APs (Fig. 4) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Thus, at this level of analysis, the zero layer Q/R interactions among SNARE proteins are not essential for SNARE function.

Effect of 12- and 24-residue insertions between the SNARE motif and transmembrane region of synaptobrevin If SNARE complexes force membranes into close proximity, the distance between the SNARE motif and transmembrane region would be expected to be critical for fusion. To test this hypothesis, we examined whether insertions of 12 or 24 residues between the SNARE motif and transmembrane region of synaptobrevin impairs its ability to mediate synaptic vesicle exocytosis (Fig. 5A). We found that synaptobrevin carrying a 12-residue insertion between SNARE motif and transmembrane region fully rescued the decline in spontaneous release in synaptobrevin-deficient neurons (Fig. 5B,C). Strikingly, however, this mutant of synaptobrevin was unable to reverse the impairment of vesicle recycling observed in synaptobrevin-deficient neurons (Fig. 5D,E), rescue the decrease in hypertonic sucrose-evoked release (Fig. 5F,G), or fully restore evoked release (Fig. 5H,I) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). In contrast to synaptobrevin 2 with a 12-residue insertion, synaptobrevin with a 24-residue insertion was unable to rescue any fusion in synaptobrevin-deficient neurons, including spontaneous fusion (Fig. 5B–I). Direct comparison of the amount of release in synaptobrevin-deficient control synapses with synaptobrevin-deficient synapses expressing exogenous synaptobrevins containing a 12-residue or 24-residue insertion showed that the 12-residue insertion was not completely inactive but approximately doubled the amount of residual evoked release in synaptobrevin-deficient neurons (Fig. 5G,I) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Figure 4. R56Q point mutation does not affect synaptobrevin 2 function. A, Diagram of the These findings suggest that the energetic constraints on sponta- point mutation at the zero layer of SNARE motif, in which the 56arginine was mutated to neous and evoked release differ and support a direct, energetic glutamine. TMR, Transmembrane region. B, Representative traces of spontaneous synaptic function of the SNARE complex in forcing the synaptic vesicle eventsrecordedfromacellinfectedwithR56Qmutantformofsynaptobrevin2.C,Frequencyof and the plasma membrane to fuse during evoked exocytosis. The spontaneoussynapticevents(nϭ12R56Qinfected;nϭ1512-insertioninfectedKOcells;nϭ small rescue effect of the 12-insertion mutant of synaptobrevin in 10 24-insertion infected KO cells). D, Average destaining curves of FM2-10-loaded synapses evoked release indicates that the distance of the SNARE motif during high potassium stimulation. E, Bar graphs depict size of recycling pool as mean Ϯ SEM ϭ ϭ from the transmembrane region is barely too long in this mutant for WT (n 16, 1146 synapses), synaptobrevin 2 KO (n 11, 688 synapses) synapses, and KO ϭ to still allow efficient fusion. synapses after infection with eCFP–synaptobrevin 2–R56Q (n 3, 126 synapses). F, A representative recording of synaptic responses to hypertonic sucrose (ϩ0.5 Osm) application in a In a parallel set of experiments, we biochemically tested synaptobrevin 2 KO culture rescued with the R56Q mutant. G, Summary graph of synaptic whether the synaptobrevin 2 with a 12-residue insertion (as well responses to hypertonic sucrose monitored in infected neurons. Abbreviations used are the as the wild-type synaptobrevin 2) could participate in SNARE same as in C. Responses are calculated as the cumulative charge transfer integrated over 1 s complex assembly. Lysates from hippocampal cultures infected interval at the peak of the response (n ϭ 6 R56Q infected). H, Representative traces from with synaptobrevin 2 and the 12-insertion mutant (Fig. 6A) were whole-cell recordings during 1 Hz field stimulations. I, Amplitudes of evoked responses (n ϭ 7 incubated with a polyclonal antibody against rat syntaxin 1 resi- WT and synaptobrevin 2 KO cells; n ϭ 11 R56Q infected cells). dues 1–264, which recognizes assembled SNARE complexes (O.-H. Shin and T. C. Su¨dhof, unpublished observations). The resulting immunoprecipitate was immunoblotted with antibod- Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion J. Neurosci., June 21, 2006 • 26(25):6668–6676 • 6673

ies against GFP, which recognizes the infected synaptobrevin 2 constructs (because an antibody raised against the N terminus of synaptobrevin 2 does not recognize these constructs because of the eCFP tag) as well as an antibody against SNAP-25 and a monoclonal antibody against syntaxin 1 (Fig. 6B). These immu- noblots clearly documented that infected wild-type synaptobrevin 2 and synaptobrevin 2 with a 12-residue insertion could form SNARE complexes. This finding supports the above premise that the distance of the SNARE motif from the transmembrane region is a major determining factor for the ineffectiveness of this mutant in rescuing evoked fusion.

Functionally inactive synaptobrevin acts as a dominant negative in wild-type neurons In a final set of experiments, we tested whether the synaptobrevin containing a 24-residue insertion that is unable to rescue the synaptobrevin knock-out phenotype alters synaptic vesicle exocytosis in wild-type synapses. When we monitored synaptic vesicle exocytosis and pool sizes in wild-type synapses expressing this construct using FM2-10 staining and destaining experiments, we found that exocytosis was severely depressed and the recycling vesicle pool size was decreased (Fig. 7). In contrast, wild-type synapses expressing either wild-type synaptobrevin or the mutant Q-synaptobrevin (both of which fully rescued the synaptobrevin deficiency phenotype in synapses) experienced no decline in fluorescent FM2-10 dye uptake and release, suggesting that only the synaptobrevin mutant that is inactive in exocytosis is dominant negative in inhibiting exocytosis. This observation further underlies the importance of the distance between the synaptobrevin SNARE motif and the transmembrane domain for SNARE core complex function in fusion. Discussion In this study, we used rescue experiments to examine how synaptobrevin acts in synaptic vesicle fusion. For this purpose, we tested mutant synaptobrevins and the closely related R-SNARE cellubrevin and used cultured hippocampal neurons from synaptobrevin-deficient mice in which we produced different proteins using a lentivirus expression system. We made five prin- cipal observations. (1) Fusion of synaptobrevin with GFP derivatives at either the N or the C terminus did not impair the ability of synaptobrevin to rescue synaptic neurotransmitter release in synaptobrevin- deficient synapses. This is significant because, in synaptotagmin 1, cytoplasmic C-terminal GFP fusions abolish function (Han et al., 2005). The fact that synaptobrevin function tolerates addition of a bulky GFP moiety at the N terminus close to the SNARE Figure 5. Membrane proximity is not an absolute requirement for spontaneous fusion. A, motif indicates that the N terminus in the SNARE complex is not Diagram of constructs used for infection. B, Representative traces of spontaneous synaptic events recorded from a cell infected with 12- and 24-insertion mutants of synaptobrevin 2. C, present in a spatially constrained environment. Frequency of spontaneous synaptic events (n ϭ 15 12-inserted infected KO cells; n ϭ 10 (2) Cellubrevin expression successfully rescued spontaneous 24-insertion infected KO cells). Note that infection with 12 insertion increased the frequency of and evoked synaptic vesicle fusion as well as efficient synaptic spontaneous synaptic events to the same level as WT. D, Average destaining curves of FM2-10- vesicle recycling. In contrast, double knock-out of cellubrevin loaded synapses during high potassium stimulation. E, Bar graphs depict size of recycling pool and synaptobrevin did not exhibit additional reduction in fusion as mean Ϯ SEM for WT (n ϭ 16, 1146 synapses), synaptobrevin 2 KO (n ϭ 11, 688 synapses) efficacy below the reduction seen in synaptobrevin knock-out synapses, and KO synapses after infection with 12 insertion (n ϭ 5, 148 synapses) and 24 alone. These data show that, although synaptobrevin and cel- insertion (n ϭ 4, 135 synapses). F, Representative traces of synaptic responses to sucrose lubrevin are functionally interchangeable, the remaining fusion monitored in infected neurons. G, Responses are calculated as the cumulative charge transfer in synaptobrevin-deficient synapses is not mediated by ϭ integrated over 1 s interval at the peak of the response [n 4 for WT and synaptobrevin 2 KO cellubrevin. cells; n ϭ 7 for 12-insertion mutant of synaptobrevin (12ins.) infected; n ϭ 3 for 24-insertion (24ins.)infectedKOcells].H,Representativetracesfromwhole-cellrecordingsduring1Hzfield (3) Exchanging the arginine in the zero layer of the SNARE stimulations.I,Amplitudesofevokedresponses(nϭ7WTandsynaptobrevin2KOcells;ϭ15 motif to glutamine (R56Q) in synaptobrevin 2 still resulted in full 12-inserted infected KO cells; n ϭ 9 24-inserted infected KO cells). Note that infection with 12 rescue of release; therefore, the four-Q combination is a func- insertion significantly increased the amplitude of evoked responses ( p Ͻ 0.05) in KO culture tional core complex alternative. but that remained far less than in WT ( p Ͻ 0.005). (4) Insertion of 12 and 24 residues between the SNARE motif 6674 • J. Neurosci., June 21, 2006 • 26(25):6668–6676 Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion

Figure 7. Dominant-negative action of the 24-insertion construct. A, High potassium induced destaining curves of FM2-10-loaded wild-type synapses infected with eCFP-tagged syn- aptobrevin2(iwSyb2),the24insertion(iw24ins),andR56Q(iwR56Q)mutants.Forcomparison, average destaining curves from synaptobrevin 2 KO and wild-type neurons are also shown. B, Bar graphs depict the size of the recycling pool as mean Ϯ SEM for WT (n ϭ 7, 400 synapses), synaptobrevin 2 KO (n ϭ 6, 364 synapses) neurons, and wild-type synapses after infection (iw24 ins, n ϭ 5, 391 synapses; iwSyb2, n ϭ 4, 409 synapses; iwR56Q, n ϭ 3, 273 synapses).

and transmembrane region of synaptobrevin 2 severely impaired the ability of synaptobrevin to mediate evoked release, demon- strating that the physical distance between the SNARE motif and transmembrane region is critical for fusion. Interestingly, however, synaptobrevin containing the 12-residue insertion completely rescued spontaneous release, although evoked release was not recovered. This result suggests that the fusion reactions me- diating evoked and spontaneous release differ. (5) Remarkably, synaptobrevin mutant with the insertion of 24 residues between the SNARE motif and the transmembrane region that was unable to rescue synaptic transmission in synaptobrevin-deficient synapses suppressed synaptic vesicle exocytosis in wild-type synapses.

Substitution of synaptobrevin function by cellubrevin SNAREs were suggested as candidates for the target specificity of vesicular transport (Sollner et al., 1993). Later findings, however, challenged this view. Transgenic rescue experiments in Drosoph- ila (Bhattacharya et al., 2002) argued for a functional inter- changeability between nonsynaptic and synaptic synaptobrevin isoforms in Drosophila. Recently, cellubrevin was shown to be responsible for the remaining release observed in synaptobrevin- deficient chromaffin cells (Borisovska et al., 2005). We observed complete rescue of synaptic neurotransmitter release when cellubrevin was expressed in synaptobrevin-deficient neurons (Fig. 2). At the same time, we found that additional deletion of cellubrevin in synaptobrevin-deficient neurons did not further aggravate the impairment in exocytosis observed in these neurons (Fig. 3), consistent with our previous finding that cellubrevin is absent from hippocampal neurons (Schoch et al., 2001). We con- clude that there is clear redundancy between the cellubrevin and synaptobrevin R-SNAREs role in fusion, despite their differences Figure 6. Infected wild-type and 12-insertion mutant of synaptobrevin 2 engage in assem- bledSNAREcomplexes.A,Cellsfromeachsix-wellplatewereharvestedusing1mlofbuffer,and in N-terminal and C-terminal sequences, suggesting that these proteins were extracted using 1% Triton X-100. A total of 50 ␮l of cell lysate was used for isoforms evolved as a means of diversifying expression patterns SDS-PAGE and Naphto blue black staining. B, A total of 10 ␮l of rabbit serum raised against rat but not of diversifying SNARE functions. syntaxin1(U6251)and20␮lofproteinA-Sepharosebeadswereaddedto1%TritonX-100cell lysate expressing mutant synaptobrevin 2 to immunoprecipitate (IP) assembled SNARE com- The “four Q configuration” in the zero layer of the SNARE plexes. After incubation, beads were washed five times, and proteins coimmunoprecipitated motif is functional with syntaxin 1 were determined by Western blotting. Shown are Western blots of proteins The lack of a major phenotype in synaptic exocytosis produced by coimmunoprecipitated with syntaxin 1. Blots were performed using the following sera or as- the Q/R substitution agrees well with previous studies in yeast cites: mouse anti-GFP, C163 from Zymed; mouse anti-Syb 2, CL69.1; mouse anti-syntaxin 1, showing that similar substitutions did not abolish SNARE func- HPC-1; and mouse anti-SNAP-25, CL71.1. tion (Katz and Brennwald, 2000; Ossig et al., 2000). Previous experiments in yeast, however, did not have the temporal resolution of synaptic exocytosis to detect changes in the kinetics or Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion J. Neurosci., June 21, 2006 • 26(25):6668–6676 • 6675 amplitude of exocytosis. Our present data thus extend the previ- albeit to different degrees (Schoch et al., 2001; Washbourne et al., ous studies by showing that the four-Q SNARE complex is not 2002). Together with the previous observations from the SNARE only functional but appears to act with a similar kinetics in exo- mutant studies, spontaneous fusion may require an alternative cytosis as the three-Q/one-R SNARE complex. This is surprising fusion complex (with a different R-SNARE) or the same complex given the tight interaction of the glutamines and arginine in the formed with less stringency (Xu et al., 1999; Melia et al., 2002). zero layer of the SNARE complex revealed in the crystal structure Stronger dependency of evoked release on the close proximity of of the complex (Sutton et al., 1998). Recent experiments with membranes is also more compatible with its strict Ca 2ϩ depen- different R–Q mutations in the zero layer of the yeast SNARE dence. Paradoxically, the mechanism of evoked release appears to proteins ykt6 and sec22 found a developmental arrest in the mu- be closely related to the evolutionally conserved exocytosis be- tants (Dilcher et al., 2001; Graf et al., 2005). These previous find- cause yeast vacuolar fusion was similarly strictly dependent on ings suggest that a subtle change in the SNARE function can lead membrane proximity and the length of R-SNAREs (McNew et to a cumulative defect in a longer time frame. These defects would al., 1999; Wang et al., 2001). be not detectable in relatively acute experimental settings such as ours. Furthermore, our system may not be optimal to detect the consequences of R-to-Q substitution in layer zero in exocytosis References Bhattacharya S, Stewart BA, Niemeyer BA, Burgess RW, McCabe BD, Lin P, because synaptic vesicle fusion is less sensitive to intracellular Boulianne G, O’Kane CJ, Schwarz TL (2002) Members of the fusion steps within a short time frame. synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter Differential requirement of membrane proximity in release and cell viability. Proc Natl Acad Sci USA 99:13867–13872. ,Ca 2؉-evoked and spontaneous synaptic release Borisovska M, Zhao Y, Tsytsyura Y, Glyvuk N, Takamori S, Matti U, Rettig J It is assumed that the assembly of SNARE complexes provides the Sudhof T, Bruns D (2005) v-SNAREs control exocytosis of vesicles from priming to fusion. EMBO J 24:2114–2126. energy for membrane fusion (Rothman, 1994; Rothman and Dea´k F, Schoch S, Liu X, Sudhof TC, Kavalali ET (2004) Synaptobrevin is Warren, 1994). Our result with the 12- and 24-residue insertion essential for fast synaptic-vesicle endocytosis. Nat Cell Biol 6:1102–1108. motif constructs provides support for the notion that SNARE Dilcher M, Kohler B, von Mollard GF (2001) Genetic interactions with the complex assembly drives approximation of membranes close to- yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6. gether. Moreover, the observation that the 24-residue insertion J Biol Chem 276:34537–34544. mutant of synaptobrevin impaired fusion in wild-type synapses Dittgen T, Nimmerjahn A, Komai S, Licznerski P, Waters J, Margrie TW, suggests that any inactive synaptobrevin, when expressed in a Helmchen F, Denk W, Brecht M, Osten P (2004) Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophys- wild-type synapse, may impair synaptobrevin function. This re- iological monitoring in vivo. Proc Natl Acad Sci USA 101:18206–18211. sult provides a possible explanation for the inactivation of synap- Fasshauer D, Sutton RB, Brunger AT, Jahn R (1998) Conserved structural tic exocytosis in wild-type synapses in which a synaptobrevin features of the synaptic fusion complex: SNARE proteins reclassified as Q- fusion protein is induced to multimerize by addition of a chem- and R-SNAREs. Proc Natl Acad Sci USA 95:15781–15786. ical dimerizer (Karpova et al., 2005). It is likely that, under these Graf CT, Riedel D, Schmitt HD, Jahn R (2005) Identification of functionally conditions, the multimerized synaptobrevin is inactivated, interacting SNAREs by using complementary substitutions in the conserved “0” layer. Mol Biol Cell 16:2263–2274. thereby having the same effect as the 24-residue insertion mutant Han W, Rhee JS, Maximov A, Lin W, Hammer RE, Rosenmund C, Sudhof TC described here. (2005) C-terminal ECFP fusion impairs synaptotagmin 1 function: Arguably the most striking finding of our experiments is that crowding out synaptotagmin 1. J Biol Chem 280:5089–5100. separating the SNARE motif and transmembrane region of syn- Hanson PI, Roth R, Morisaki H, Jahn R, Heuser JE (1997) Structure and aptobrevin by sequence insertions has distinct effects on evoked conformational changes in NSF and its membrane receptor complexes and spontaneous fusion. The dramatic effect of the 12-residue visualized by quick-freeze/deep-etch electron microscopy. Cell 90:523–535. insertion on evoked exocytosis confirms, to our knowledge for Jahn R, Lang T, Sudhof TC (2003) Membrane fusion. Cell 112:519–533. the first time for a physiological fusion reaction followed in real Karpova AY, Tervo DGR, Gray NW, Svoboda K (2005) Rapid and reversible time, that tight coupling of the SNARE complex to the trans- chemical inactivation of synaptic transmission in genetically targeted membrane region is essential for exocytosis. Curiously, however, neurons. Neuron 48:727–735. the same insertion had no effect on the ability of synaptobrevin to Katz L, Brennwald P (2000) Testing the 3Q:1R “rule”: mutational analysis of rescue spontaneous release (Fig. 5B,C). This finding argues the ionic “zero” layer in the yeast exocytic SNARE complex reveals no against the traditional notion that spontaneous release events requirement for arginine. Mol Biol Cell 11:3849–3858. Link E, Edelmann L, Chou JH, Binz T, Yamasaki S, Eisel U, Baumert M, arise from the random low-probability exocytosis of docked and Sudhof TC, Niemann H, Jahn R (1992) Tetanus toxin action: inhibition fully primed vesicles in the readily releasable pool. According to of neurotransmitter release linked to synaptobrevin proteolysis. Biochem this view, spontaneous fusion should possess the same structural Biophys Res Commun 189:1017–1023. requirements as evoked fusion. Our finding cannot be ascribed to McMahon HT, Ushkaryov YA, Edelmann L, Link E, Binz T, Niemann H, Jahn a selective effect of synaptobrevin 2 in Ca 2ϩ dependence of R, Sudhof TC (1993) Cellubrevin is a ubiquitous tetanus-toxin substrate evoked fusion (Sakaba et al., 2005; Young, 2005) because synap- homologous to a putative synaptic vesicle fusion protein. Nature 364:346–349. tobrevin carrying the 12-residue insertion was also essentially McNew JA, Weber T, Engelman DM, Sollner TH, Rothman JE (1999) The unable to rescue hypertonic sucrose-evoked fusion, which is length of the flexible SNAREpin juxtamembrane region is a critical deter- 2ϩ Ca independent (Rosenmund and Stevens, 1996). minant of SNARE-dependent fusion. Mol Cell 4:415–421. Our findings are consistent with the recent observation that Melia TJ, Weber T, McNew JA, Fisher LE, Johnston RJ, Parlati F, Mahal LK, the spontaneous neurotransmission originates, at least in part, Sollner TH, Rothman JE (2002) Regulation of membrane fusion by the from a different vesicle population than evoked neurotransmis- membrane-proximal coil of the t-SNARE during zippering of SNARE- sion (Sara et al., 2005). This recent study raised the question pins. J Cell Biol 158:929–940. Ossig R, Schmitt HD, de Groot B, Riedel D, Keranen S, Ronne H, Grubmuller whether the release machinery for spontaneous fusion is identical H, Jahn R (2000) Exocytosis requires asymmetry in the central layer of to the one required for evoked fusion. Clearly, both forms of the SNARE complex. EMBO J 19:6000–6010. fusion are impaired in the absence of the same SNARE molecules, Parlati F, McNew JA, Fukuda R, Miller R, Sollner TH, Rothman JE (2000) 6676 • J. Neurosci., June 21, 2006 • 26(25):6668–6676 Dea´k et al. • Synaptobrevin Function in Synaptic Vesicle Fusion

Topological restriction of SNARE-dependent membrane fusion. Nature Neher E (2003) Differential control of the releasable vesicle pools by 407:194–198. SNAP-25 splice variants and SNAP-23. Cell 114:75–86. Rosenmund C, Stevens CF (1996) Definition of the readily releasable pool Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a of vesicles at hippocampal synapses. Neuron 16:1197–1207. SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Na- Rothman JE (1994) Mechanisms of intracellular protein transport. Nature ture 395:347–353. 372:55–63. Wang Y, Dulubova I, Rizo J, Su¨dhof TC (2001) Functional analysis of con- Rothman JE, Warren G (1994) Implications of the SNARE hypothesis for served structural elements in yeast syntaxin Vam3p. J Biol Chem intracellular membrane topology and dynamics. Curr Biol 4:220–233. 276:28598–28605. Sakaba T, Stein A, Jahn R, Neher E (2005) Distinct kinetic changes in neu- Washbourne P, Thompson PM, Carta M, Costa ET, Mathews JR, Lopez- rotransmitter release after SNARE protein cleavage. Science 309:491–494. Bendito G, Molnar Z, Becher MW, Valenzuela CF, Partridge LD, Wilson Sara Y, Virmani T, Dea´k F, Liu X, Kavalali ET (2005) An isolated pool of MC (2002) Genetic ablation of the t-SNARE SNAP-25 distinguishes vesicles recycles at rest and drives spontaneous neurotransmission. Neu- mechanisms of neuroexocytosis. Nat Neurosci 5:19–26. ron 45:563–573. Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, Das- Gupta BR, Montecucco C (1992) Tetanus and botulinum-B neurotox- Sollner TH, Rothman JE (1998) SNAREpins: minimal machinery for ins block neurotransmitter release by proteolytic cleavage of synaptobre- membrane fusion. Cell 92:759–772. vin. Nature 359:832–835. Xu T, Rammner B, Margittai M, Artalejo AR, Neher E, Jahn R (1999) Inhi- Schoch S, Dea´k F, Konigstorfer A, Mozhayeva M, Sara Y, Sudhof TC, Kavalali bition of SNARE complex assembly differentially affects kinetic compo- ET (2001) SNARE function analyzed in synaptobrevin/VAMP knock- nents of exocytosis. Cell 99:713–722. out mice. Science 294:1117–1122. Yang C, Mora S, Ryder JW, Coker KJ, Hansen P, Allen LA, Pessin JE (2001) Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos VAMP3 null mice display normal constitutive, insulin- and exercise- S, Tempst P, Rothman JE (1993) SNAP receptors implicated in vesicle regulated vesicle trafficking. Mol Cell Biol 21:1573–1580. targeting and fusion. Nature 362:318–324. Young Jr SM (2005) Proteolysis of SNARE proteins alters facilitation and Sorensen JB, Nagy G, Varoqueaux F, Nehring RB, Brose N, Wilson MC, depression in a specific way. Proc Natl Acad Sci USA 102:2614–2619.